Insects and other pests cost farmers billions of dollars annually in crop losses and in the expense of keeping these pests tinder control. The losses caused by pests in agricultural production environments include decrease in crop yield, reduced crop quality, and increased harvesting costs.
Coleopterans are an important group of agricultural pests which cause a very large amount of damage each year. Examples of coleopteran pests include alfalfa weevils and corn rootworm.
The alfalfa weevil, Hyperca postica, and the closely related Egyptian alfalfa weevil, Hyperca brunneipennis, are the most important insect pests of alfalfa grown in the United States, with 2.9 million acres infested in 1984. An annual sum of 20 million dollars is spent to control these pests. The Egyptian alfalfa weevil is the predominant species in the southwestern U.S., where it undergoes aestivation (i.e., hibernation) during the hot summer months. In all other respects, it is identical to the alfalfa weevil, which predominates throughout the rest of the U.S.
The larval stage is the most damaging in the weevil life cycle. By feeding at the alfalfa plant's growing tips, the larvae cause skeletonization of leaves, stunting, reduced plant growth, and, ultimately, reductions in yield. Severe infestations can ruin an entire cutting of hay. The adults, also foliar feeders, cause additional, but less significant, damage.
Approximately 9.3 million acres of U.S. corn are infested with corn rootworm species complex each year. The corn rootworm species complex includes the northern corn rootworm, Diabrotica barberi, the southern corn rootworm, D. undecimpunctata howardi, and the western corn rootworm, D. virgifera virgifera. The soil-dwelling larvae of these Diabrotica species feed on the root of the corn plant, causing lodging. Lodging eventually reduces corn yield and often results in death of the plant. By feeding on cornsilks, the adult beetles reduce pollination and, therefore, detrimentally effect the yield of corn per plant. In addition, members of the genus Diabrotica attack cucurbit crops (cucumbers, melons, squash. etc.) and many vegetable and field crops in commercial production as well as those being grown in home gardens.
Control of corn rootworm has been partially addressed by cultivation methods, such as crop rotation and the application of high phosphate levels to stimulate the growth of an adventitious root system. In addition, an emerging two-year diapause (or overwintering) trait of Northern corn rootworms is disrupting crop rotation in some areas. However, chemical insecticides are relied upon most heavily to guarantee the desired level of control. Insecticides are either banded onto or incorporated into the soil. The major problem associated with the use of chemical insecticides is the development of resistance among the treated insect populations.
Over $250 million worth of insecticides are applied annually to control corn rootworms alone in the United States. Even with insecticide use, rootworms cause over $750 million worth of crop damage each year, making them the most serious corn insect pest in the Midwest.
Damage to plants caused by nematodes is also a prevalent and serious economic problem. Nematodes cause wide-spread and serious damage in many plant species. Many genera of nematodes are known to cause such damage. Plant-parasitic nematodes include members of the Phylum Nematoda, Orders Tylenchida and Dorylaimide. In the Order Tylenchida, the plant-parasitic nematodes are found in two Super Families: Tylenchoidea and Criconematoidea. There are more than 100,000 described species of nematodes.
Chemical pesticides have provided an effective method of pest control; however, the public has become concerned about the amount of residual chemicals that might be found in food, ground water, and the environment. Stringent new restrictions on the use of pesticides and the elimination of some effective pesticides form the marketplace could limit economical and effective options for controlling costly pests. Thus, there is an urgent need to identify pest control methods and compositions which are not harmful to the environment.
Nematicides routinely used for control of plant-parasitic nematodes are rapidly being pulled from the market as concern for environmental safety increases. In the year 2001, Methyl Bromide, a mainstay in the control of such parasites, will no longer be marketed in the United States. Therefore, less harmful control agents are clearly needed.
The use of chemical pesticides to control corn rootworm and other coleopteran pests, as well as nematodes, has several drawbacks. Pesticide use often raises environmental concerns such as contamination of soil and of both surface and underground water supplies. Working with pesticides may also pose hazards to the persons applying them.
The regular use of chemical pesticides for the control of unwanted organisms can select for chemical resistant strains. Chemical resistance occurs in many species of economically important insects and has also occurred in nematodes of sheep, goats, and horses. The regular use of chemical toxins to control unwanted organisms can select for drug-resistant strains. This has occurred in many species of economically important insects and has also occurred in nematodes of sheep, goats, and horses. For example, an accepted methodology for control of nematodes has centered around the drug benzimidazole and its congeners. The use of these drugs on a wide scale has led to many instances of resistance among nematode populations (Prichard, R. K. et al. [1980] "The problem of anthelmintic resistance in nematodes," Austr. Vet. J. 56:239-251; Coles, G. C. [1986] "Anthelmintic resistance in sheep," In Veterinary Clinics of North America: Food Animal Practice, Vol 2:4-432 [Herd, R. P., eds.] W. B. Saunders, New York). There are more than 100,000 described species of nematodes. The development of pesticide resistance necessitates a continuing search for new control agents having different modes of action.
At the present time there is a need to have more effective means to control the many coleopterans and nematodes that cause considerable damage to susceptible hosts and crops. Advantageously, such effective means would employ specific biological agents.
The soil microbe Bacillus thuringiensis (B.t.) is a Gram-positive, spore-forming bacterium characterized by parasporal crystalline protein inclusions. These inclusions often appear microscopically as distinctively shaped crystals. The proteins can be highly toxic to pests and specific in their toxic activity. Certain B.t. toxin genes have been isolated and sequenced, and recombinant DNA-based B.t. products have been produced and approved for use. In addition, with the use of genetic engineering techniques, new approaches for delivering these B.t. endotoxins to agricultural environments are under development, including the use of plants genetically engineered with endotoxin genes for insect resistance and the use of stabilized intact microbial cells as B.t. endotoxin delivery vehicles (Gaertner, F. H., L. Kim [1988] TIBTECH 6:54-57). Thus, isolated B.t. endotoxin genes are becoming commercially valuable.
Until fairly recently, commercial use of B.t. pesticides has been largely restricted to a narrow range of lepidopteran (caterpillar) pests. Preparations of the spores and crystals of B. thuringiensis subsp. kurstaki have been used for many years as commercial insecticides for lepidopteran pests. For example, B. thuringiensis var. kurstaki HD-1 produces a crystalline .delta.-endotoxin which is toxic to the larvae of a number of lepidopteran insects.
In recent years, however, investigators have discovered B.t. pesticides with specificities for a much broader range of pests. For example, other species of B.t., namely israelensis and morrisoni (a.k.a. tenebrionis, a.k.a. B.t. M- 7, a.k.a. B.t. san diego), have been used commercially to control insects of the orders Diptera and Coleoptera, respectively (Gaertner, F. H. [1989] "Cellular Delivery Systems for Insecticidal Proteins: Living and Non-Living Microorganisms," in Controlled Delivery of Crop Protection Agents, R. M. Wilkins, ed., Taylor and Francis. New York and London, 1990, pp. 245-255.). See also Couch, T. L. (1980) "Mosquito Pathogenicity of Bacillus thuringiensis var. israelensis," Developments in Industrial Microbiology 22:61-76; and Beegle, C. C., (1978) "Use of Entomogenous Bacteria in Agroecosystems," Developments in Industrial Microbiology 20:97-104. Krieg, A., A. M. Huger, G. A. Langenbruch, W. Schnetter (1983) Z. ang. Ent. 96:500-508, describe Bacillus thuringiensis var. tenebrionis, which is reportedly active against two beetles in the order Coleoptera. These are the Colorado potato beetle, Leptinotarsa decemlineata, and Agelastica alni.
More recently, new subspecies of B.t. have been identified, and genes responsible for active .delta.-endotoxin proteins have been isolated (Hofte, H., H. R. Whiteley [1989] Microbiological Reviews 52(2): 242-255). Hofte and Whiteley classified B.t. crystal protein genes into four major classes. The classes were CryI (Lepidoptera-specific), CryII (Lepidoptera- and Diptera-specific), CryIII (Coleoptera-specific), and CryIV (Diptera-specific). The discovery of strains specifically toxic to other pests has been reported (Feitelson, J. S., J. Payne, L. Kim [1992] Bio/Technology 10:271-275). CryV was proposed to designate a class of toxin genes that are nematode-specific. Other classes of B.t. genes have now been identified.
The 1989 nomenclature and classification scheme of Hofte and Whiteley for crystal proteins was based on both the deduced amino acid sequence and the host range of the toxin. That system was adapted to cover 14 different types of toxin genes which were divided into five major classes. As more toxin genes were discovered, that system started to become unworkable, as genes with similar sequences were found to have significantly different insecticidal specificities. The number of sequenced Bacillus thuringiensis crystal protein genes currently stands at about 50. A revised nomenclature scheme has been proposed which is based solely on amino acid identity (Crickmore et al. [1996] Society for Invertebrate Pathology, 29th Annual Meeting, IIIrd International Colloquium on Bacillus thuringiensis, University of Cordoba, Cordoba, Spain, September 1-6, abstract). The mnemonic "cry" has been retained for all of the toxin genes except cytA and cytB, which remain a separate class. Roman numerals have been exchanged for Arabic numerals in the primary rank, and the parentheses in the tertiary rank have been removed. Many of the original names have been retained, with the noted exceptions, although a number have been reclassified.
The cloning and expression of a B.t. crystal protein gene in Escherichia coli has been described in the published literature (Schnepf, H. E., H. R. Whiteley (1981) Proc. Natl. Acad. Sci. USA 78:2893-2897). U.S. Pat. Nos. 4,448,885 and 4,467,036 both disclose the expression of B.t. crystal protein in E. coli. U.S. Pat. Nos. 4,990,332; 5,039,332; 5,126,133; 5,164,180; 5,169,629 and 5,286,485 are among those which disclose B.t. toxins having activity against lepidopterans. U.S. Pat. Nos. 4,797,276 and 4,853,331 disclose B. thuringiensis strain tenebrionis which can be used to control coleopteran pests in various environments. U.S. Pat. No. 4,918,006 discloses B.t. toxins having activity against dipterans.
A small number of research articles have been published about the effects of delta endotoxins from B. thuringiensis species on the viability of nematode eggs. Bottjer, Bone and Gill, ([1985] Experimental Parasitology 60:239-244) have reported that B.t. kurstaki and B.t. Israelensis were toxic in vitro to eggs of the nematode Trichostrongylus colubriformis. In addition, 28 other B.t. strains were tested with widely variable toxicities. Ignoffo and Dropkin ([1977] J. Kans. Entoml. Soc. 50:394-398) have reported that the thermostable toxin from Bacillus thuringiensis (beta exotoxin) was active against a free-living nematode, Panagrellus redivivus (Goodey); a plant-parasitic nematode, Meloidogyne incognita (Chitwood); and a fungus-feeding nematode, Aphelenchus avena (Bastien). Beta exotoxin is a generalized cytotoxic agent with little or no specificity. Also, Ciordia and Bizzell ([1961] Jour. of Parasitology 47:41 [abstract]) gave a preliminary report on the effects of B. thuringiensis on some cattle nematodes.
U.S. Pat. Nos. 5,151,363 and 4,948,734 disclose certain isolates of B.t. which have activity against nematodes. Other U.S. Patents which disclose activity against nematodes include U.S. Pat. Nos. 5,093,120; 5,236,843; 5,262,399; 5,270,448; 5,281,530; 5,322,932; 5,350,577; 5,426,049; and 5,439,881. As a result of extensive research and investment of resources, other patents have issued for new B.t. isolates and new uses of B.t. isolates. See Feitelson et al., supra, for a review. However, the discovery of new B.t. isolates and new uses of known B.t. isolates remains an empirical, unpredictable art.
Some Bacillus thuringiensis toxins which are active against corn rootworm and other coleopterans are now known. For example, U.S. Pat. No. 4,849,217 discloses various isolates, including PS52A1 and PS86A1, as having activity against alfalfa weevils. U.S. Pat. No. 5,208,017 discloses PS86A1 as a having activity against Western corn rootworm. U.S. Pat. Nos. 5,427,786 and 5,186,934 each disclose B.t. isolates and toxins active against coleopterans. Specifically disclosed in these patents is the isolate known as PS86A1 and a coleopteran-active toxin obtainable therefrom known as 86A1. Toxin 86A1 is now also known as Cry6A (CryVIA). The wild-type Cry6A toxin is about 54-58 kDa.
A Cry6B toxin is also known. This toxin can be obtained from the PS69D1 isolate. The full length Cry6A and Cry6B toxins are known to have activity against nematodes. The following U.S. Patents disclose, in part, the PS69D1 isolate as having activity against nematodes: U.S. Pat. Nos. 4,948,734; 5,093,120; 5,262,399; and 5,439,881.
A generic formula for the amino acid sequence of CryVI toxins has been disclosed in WO 92/19739, which also teaches that the full length toxin has activity against nematodes. The PS52A1 and PS69D1 isolates are disclosed therein. U.S. Pat. Nos. 5,262,159 and 5,468,636 also disclose a generic formula for toxins having activity against aphids.
Although the Cry6A toxin was known to inhibit the growth of certain coleopterans, it was not previously known that this toxin could be activated by truncation to yield a toxin that is lethal to coleopterans, such as the western corn rootworm. In addition, there was no suggestion that the truncated Cry6A would be active against nematodes.
Some previous examples of truncations to other B.t. toxins are known in the art. For example, the P2 (Cry2) toxins (Nicholles, E. N., W. Ahmad. D. J. Ellar [1989] J. Bact. 171:5141-5147) exist as 61-63 kDa proteins. Proteolysis trims about 5 kDa off, leaving 56-58 kDa proteins. However, toxicity either remained unchanged or was worse by a factor of 10. Furthermore, these proteins share no significant homology with Cry6 toxins. Other articles which address certain aspects of the activity and/or function of portions of B.t. toxins include Adang, M. J., M. J. Staver, T. A. Rocheleau et al. (1985) Gene 36:289-300; Wabiko, H., K. C. Raymond, L. A. Bulla, Jr. (1986) DNA 5:305-314 (Medline 863000920); Schnepf, H. E., H. R. Whiteley (1985)J. Biol. Chem. 260:6273-6280; U.S. Pat. Nos. 5,468,636; 5,236,843; and EP 0462721.
The use of truncation to obtain activated Cry6A toxins as described below is completely new to the B.t. art.